Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that act as ligand-dependent transcription factors (Kersten et al 2000). PPARs are also ligand-inducible transcription factors belonging to the steroid, thyroid and retinoid receptor superfamily and also termed nuclear hormone receptors (Desvergne and Wahli 1999; Straus and Glass 2001). Nuclear receptors directly bind to DNA and regulate gene expression through transcriptional co-activation (Nolte et al. 1998; Berger and Moller 2002; Castrillo and Tontonoz 2004). The PPAR subfamily is comprised of three isoforms: PPAR-α, PPAR-β/δ and PPAR-γ, and these isoforms share structural homology in various species (Desvergne and Wahli 1999; Bishop-Bailey 2000; Buchan and Hassall 2000; Straus and Glass 2001). PPARα, β, and γ are the three commonly known PPAR isotypes. PPARα is predominantly expressed in the liver, kidney, muscle, adipose, and heart, whereas PPARβ is found in the brain, adipose, and skin, and PPARγ is expressed ubiquitously (Bensinger and Tontonoz 2008). These transcription factors have been linked to lipid transport, metabolism, and inflammation pathways (Bensinger and Tontonoz 2008). Because of this, synthetic PPAR agonists have been generated as therapeutic agents for the treatment of diabetes and metabolic diseases (Schulman 2010; Wang 2010).
PPAR are activated by small, lipophilic compounds and form heterodimers with the retinoid X receptor-a (RXR) in the cytoplasm for full activation (van Neerven and Mey 2007). After activation the PPAR/RXR heterodimer binds to the specific DNA sequence (peroxisome proliferator response element; PPRE) on the promoter region of PPAR target genes (Desvergne and Wahli 1999; Qi et al. 2000) to modulate transcriptional activity. Specific binding of PPAR on DNA sequences leads to activation of hundreds of gene cascades involved in several biological processes (Qi et al. 2000). In the absence of ligands, PPAR and RXR heterodimers bind to co-repressor complexes and suppress gene transcription (Ziouzenkova and Plutzky 2008).
While, binding of PPAR with specific ligands leads to release of co-repressors from heterodimers and recruitment of co-activators, followed by activation of the basal transcriptional machinery (Kamei et al. 1996; Desvergne and Wahli 1999; Straus and Glass 2001), dietary lipids and their metabolites, fatty acids and eicosanoids are the natural ligands for PPAR. However, these receptors are also activated by synthetic ligands such as thiazolidinediones, fibrates, W501516 and L-165041 (Desvergne and Wahli 1999; Straus and Glass 2007). Several non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, fenoprofen and indomethacin also activate PPARα and PPAR-γ (Lehmann et al. 1997). It was postulated that the anti-inflammatory actions of these drugs may arise from their ability to bind to PPAR, and subsequent activation of these receptors (Jiang et al. 1998; Ricote et al. 1998; Casper et al. 2000; Heneka and Landreth 2007).
Different PPAR isoforms develop from a common PPAR gene and show tissue dependent patterns of expression during fetal development and are involved in differentiation of adipose tissue, brain, skin, liver, muscle and placenta (Desvergne and Wahli 1999; Gofflot et al. 2007). On the basis of target genes and differential localization in the tissues, these isoforms perform different pharmacological, physiological and biological functions and exhibit different ligand specificities (Desvergne and Wahli 1999). PPAR-α is activated by natural fatty acids and synthetic fibrate ligands and regulates metabolism of lipid and apolipoproteins. PPAR-γ is involved in regulation of adipocyte differentiation (adipogenesis), glucose metabolism, insulin sensitivity and cell growth and is activated by natural ligands as well as synthetic glitazone ligands, while, PPAR-β/δ regulates lipid and glucose metabolism.
PPARs can affect metabolism and inflammation in the central nervous system (Heneka and Landreth 2007), suggesting that they can play a role in the pathogenesis of neurodegenerative diseases. PPAR agonists increase oxidative phosphorylation capacity in mouse and human cells (Bastin et al 2008; Hondares et al 2006; Wenz et al 2010), and enhance mitochondrial biogenesis.
Prior reports have demonstrated beneficial effects of PPARγ agonists, such as thiazolidinediones (TZD, also called glitazones) (Kaundal and Sharma 2010), in models of stroke (Culman et al 2007) and Alzheimer's disease (Heneka et al 2005; Jiang et al 2008; Nicolakakis et al 2008; Nicolakakis and Hamel 2010). Fibrates, such as fenofibrate (Rakhshandehroo 2010), are another class of PPAR agonists (Abourbih et al 2009; Munigoti and Rees 2011; Staels et al 2008) that primarily target the PPARα pathway. Like TZD, fenofibrate has demonstrated promising protective effects in models of neurodegenerative diseases, including Parkinson's disease (Kreisler et al 2010), and brain injury (Chen et al 2007). Interestingly, the neuroprotective effects of PPAR agonists seem to occur through a common mechanism involving the reduction of oxidative stress and inflammation (Heneka et al 2005; Jiang et al 2008; Nicolakakis et al 2008; Nicolakakis and Hamel 2010; Chen et al 2007).
Bezafibrate is a member of the fibrate family that predominantly activates PPARα, but can also act on PPARβ and γ (Tenenbaum et al 2005). It can therefore be considered a pan-PPAR agonist. Recently, the administration of bezafibrate was shown to increase PGC-1α expression, mitochondrial DNA and ATP levels; and to increase life span and delay myopathy in a COX-10 subunit deficient mouse model of mitochondrial myopathy (Wenz et al 2008). Bezafibrate enhances lipid metabolism and oxidative capacity (Tenenbaum et al 2005; Bastin et al 2008). Bezafibrate is an effective cholesterol lowering drug which is used to lower cholesterol and triglycerides and increase high density lipoprotein (HDL).
Huntington's disease (HD), is a fatal, dominantly inherited progressive neurodegenerative disease, caused by an abnormal CAG repeat expansion in the huntingtin (htt) gene. The disease is characterized by progressive motor impairment, personality changes, psychiatric illness and gradual intellectual decline, leading to death 15-20 years after onset (Vonsattel and DiFiglia 1998). Neuropathological analysis shows a preferential and progressive loss of the medium spiny neurons (MSNs) in the striatum, although cortical atrophy and degeneration of other brain regions occur in later stages of the disease (Vonsattel and DiFiglia 1998; Hayden and Kremer 2001; Zuccato et al 2010). Several transgenic mouse models exist that recapitulate the main features of HD, and which have been used for development and testing of new therapeutic interventions. Transgenic mouse models either contain htt N-terminal fragments, usually the first 1 or 2 exons of the human htt gene with the CAG expansion, or the full-length human HD gene with an expanded CAG tract. All these models share features with human HD. The most extensively studied are the R6/2 mice, which express exon-1 of the human htt gene, and which initially show behavioral and motor deficits at 6 weeks after birth. Subsequently, the phenotype of the R6/2 mice develops rapidly manifesting tremor, clasping, weight loss, diabetes, behavioral abnormalities, and reduced life span of 10-13 weeks (Mangiarini et al 1996; Menalled and Chesselet 2002).
Transcriptional dysregulation, protein aggregation, mitochondrial dysfunction and enhanced oxidative stress have been implicated in the disease pathogenesis. A critical role of peroxisome proliferator activated receptor (PPAR)-γ-coactivator 1α (PGC-1α), a transcriptional master co-regulator of mitochondrial biogenesis, metabolism and anti-oxidant defenses, has been identified in HD. Interest in the role of PGC-1α in HD pathogenesis initially came from studies of PGC-1α knockout mice (PGC-1αKO), that display neurodegeneration in the striatum, which is also the brain region most affected in HD (Lin et al. 2004; Leone et al. 2005). PGC-1α also plays a role in the suppression of oxidative stress, and it induces mitochondrial uncoupling proteins and antioxidant enzymes, including copper/zinc superoxide dismutase (SOD1), manganese SOD (SOD2), and glutathione peroxidase (Gpx-1) (St-Pierre et al 2006). Oxidative damage is a well characterized feature which is documented in plasma of HD patients, HD postmortem brain tissue, and in HD transgenic mice (Browne and Beal 2006; Hersch et al. 2006).
Using striata from human HD patients, striata from HD knock-in mice and the STHdhQ111 cell-based HD model, Cui et al. (Cui et al. 2006) showed marked reductions in mRNA expression of PGC-1α, and interference of mutant htt with the CREB/TAF4 complex was shown to be instrumental in this reduction. Down-regulation of PGC-1α significantly worsened the behavioral and neuropathological abnormalities in a PGC-1α knock-out HD knock-in mouse model (PGC-1αKO/KI). Administration of a lentiviral vector expressing PGC-1α into the striatum of R6/2 mice, was neuroprotective in that it increased the mean neuronal volume of medium spiny neurons (Cui et al. 2006). Caudate nucleus microarray expression data from human HD patients showed significant reductions in 24 out of 26 PGC-1α target genes (Weydt et al 2006). These authors also found reduced PGC-1α mRNA expression in striata of the N171-82Q transgenic mouse model of HD.
Subsequent studies were carried out, which showed that the ability to upregulate PGC-1α in response to an energetic stress, produced by administration of the creatine analogue, guanidinopropionic acid, was markedly impaired in HD transgenic mice (Chaturvedi et al. 2009; Chaturvedi et al. 2010). PGC-1α plays a critical role in mitochondrial biogenesis in muscle, and in influencing whether muscle contains slow-twitch oxidative or fast-twitch glycolytic fibers (Lin et al. 2002). Impaired generation of ATP in muscle and a myopathy occurs in gene-positive individuals at risk for HD, HD patients and HD transgenic mice (Gizatullina et al 2006; Kosinski et al. 2007; Turner et al 2007). Impaired PGC-1α activity was observed in muscle of HD transgenic mice, and in myoblasts and muscle biopsies from HD patients (Chaturvedi et al 2010). A pathologic grade-dependent significant reduction in numbers of mitochondria in striatal spiny neurons, which correlated with reductions in PGC-1α and the mitochondrial transcription factor a (Tfam) was also showed (Kim et al 2010). Sequence variation in the PGC-1α gene modifies the age of onset of HD (Weydt et al 2009; Taherzadeh-Fard et al 2009). Stimulation of extrasynaptic NMDA receptors, which is detrimental, impairs the PGC-1α cascade in HD mice (Okamoto et al 2009). Impaired PGC-1α was shown to correlate with lipid accumulation in brown adipose tissue of HD transgenic mice (Phan et al 2009). These findings in concert, strongly implicate reduced expression of PGC-1α in HD pathogenesis. If impaired PGC-1α transcriptional activity is playing an important role in HD pathogenesis, then pharmacologic agents such as bezafibrate which increase its levels and activity might be beneficial.
With respect to tauopathies, although previous reports have shown that PPAR agonists can reduce amyloid-β (Aβ), studies to clarify the role of PPARs in Alzheimer's disease are necessary. In particular, the relationship between PPARs and the protein tau should be explored. Presently, whether a pan-PPAR agonist can be beneficial in models of Alzheimer's disease and tauopathy, specifically, the effect of bezafibrate administration in P301S mice was investigated
Increased phosphorylation and accumulation of tau within neurons are important hallmarks of Alzheimer's disease and tauopathies. P301S transgenic mice, which carry the mutated human tau gene (P301S mutation), develop progressive tau pathology, behavioral (Scattoni et al 2010) and synaptic deficits (Yoshiyama et al 2007), and microglial activation (Yoshiyama et al 2007; Bellucci et al 2004).
There is a large body of evidence demonstrating the importance of PPARs in lipid metabolism, energy metabolism, and inflammation. Several groups have investigated the role of PPARs in the central nervous system and their effects in models of neurodegeneration (Heneka and Landreth 2007).
PPARγ agonists such as pioglitazones and rosiglitazones have been widely used in models of neurodegenerative diseases. Previously, it was reported that administration of pioglitazone extended survival, and attenuated neuronal loss, gliosis, and oxidative stress in a mouse model of amyotrophic lateral sclerosis (ALS) (Kiaei et al 2005). Similar results were found in the transgenic mouse models of Alzheimer's disease (AD) (Landreth 2007). These drugs had protective effects in transgenic mice modeling AD by reducing Aβ levels, inflammation, (Heneka et al 2005) and cerebrovascular dysfunction (Nicolakakis et al 2008). In addition to behavioral improvement (Escribano et al 2009), rosiglitazone also enhanced mitochondrial biogenesis (Strum et al 2007). Upregulation of PPARγ in neuroblastoma cells transfected with the human amyloid precursor gene (APP) was neuroprotective as evidenced by a reduction of H2O2-induced cell death and Aβ secretion (d'Abramo et al 2005). Other PPAR agonists have been tested as potential therapeutic agents for the treatment of neurodegenerative diseases. PPARα agonists, such as fenofibrate, show promising effects in mouse models of Parkinson's disease (PD) (Kreisler et al 2010) and brain injury (Besson et al 2005; Deplanque et al. 2003). In the latter, data showed that the neuroprotection was due to elevated antioxidant enzyme activities and reduced markers of inflammation. In primary neuronal cells, administration of Wy-14.463, a PPARα agonist, reduced Aβ-induced cell death, reactive oxygen species (ROS) production, and elevated calcium level by upregulating mitochondrial antioxidant enzymes (Santos et al 2005).
Tauopathies are a class of neurodegenerative diseases or effects of CNS trauma characterized by a pathological aggregation of tau protein in the human brain. The best known of these illnesses is Alzheimer's disease (AD), where tau protein is deposited within neurons in the form of neurofibrillary tangles (NFTs). Tangles are formed by hyperphosphorylation of a microtubule-associated protein known as tau, causing it to aggregate in an insoluble form (These aggregations of hyperphosphorylated tau protein are also referred to as PHF, or “paired helical filaments”). Other tauopathies include: Progressive supranuclear palsy; Dementia pugilistica (chronic traumatic encephalopathy); traumatic encephalopathy; Frontotemporal dementia and parkinsonism linked to chromosome 17; Lytico-Bodig disease (Parkinson-dementia complex of Guam); Tangle-predominant dementia; Ganglioglioma; gangliocytoma; Meningioangiomatosis; Subacute sclerosing panencephalitis; lead encephalopathy; tuberous sclerosis; Hallervorden-Spatz disease; lipofuscinosis; Pick's disease; corticobasal degeneration; Argyrophilic grain disease (AGD); corticobasal degeneration; Frontotemporal dementia; and Frontotemporal lobar degeneration. The non-Alzheimer's tauopathies are grouped together as members of “Pick's complex”. For the purposes of this patent application, Parkinson's disease is not a tauopathy.
Although a number of studies have shown that agonists targeting individual PPARs have neuroprotective efficacy, there have not previously been studies of pan-PPAR agonists in transgenic mouse models of neurodegenerative diseases. Other approaches to achieving pan-PPAR effects include utilizing combinations of agonists that act either at the individual PPAR subtypes or at two PPAR subtypes. Examples of the latter include the glitazars, which operate as agonists of PPAR α and γ, and include aleglitazar, muraglitazar and tesaglitazar (Staels 2002). PPAR gamma agonists include the thiazolidinediones, also known as glitazones, which include rosiglitazone, pioglitazone, and troglitazone which are marketed drugs, as well as experimental agents MCC-555, rivoglitazone, and ciglitazone. NSAIDS such as ibuprofen and naproxen activate PPARγ (Dill et al 2010). Other PPARγ agonists include GW1929, azelacyl PAF, and BUT.13. PPAR alpha agonists include fibrates other than bezafibrate, such as CP-751461, CP868388, GW7647 and WY-14643. PPAR-beta/delta agonists include GW0742 and L165,041.
There is still a need to develop an effective potent therapeutic method for the treatment of neurodegenerative diseases, particularly for Huntington's disease and/or tauopathies, since there is as yet no cure for these disorders, and no therapy to delay the onset of symptoms.